title: A single point mutation in ricin A-chain increases toxin degradation and inhibits EDEM1-dependent ER retrotranslocation.
abstract: Ricin is a potent plant cytotoxin composed of an A-chain [RTA (ricin A-chain)] connected by a disulfide bond to a cell binding lectin B-chain [RTB (ricin B-chain)]. After endocytic uptake, the toxin is transported retrogradely to the ER (endoplasmic reticulum) from where enzymatically active RTA is translocated to the cytosol. This transport is promoted by the EDEM1 (ER degradation-enhancing α-mannosidase I-like protein 1), which is also responsible for directing aberrant proteins for ERAD (ER-associated protein degradation). RTA contains a 12-residue hydrophobic C-terminal region that becomes exposed after reduction of ricin in the ER. This region, especially Pro250, plays a crucial role in ricin cytotoxicity. In the present study, we introduced a point mutation [P250A (substitution of Pro250 with alanine)] in the hydrophobic region of RTA to study the intracellular transport of the modified toxin. The introduced mutation alters the secondary structure of RTA into a more helical structure. Mutation P250A increases endosomal-lysosomal degradation of the toxin, as well as reducing its transport from the ER to the cytosol. Transport of modified RTA to the cytosol, in contrast to wild-type RTA, appears to be EDEM1-independent. Importantly, the interaction between EDEM1 and RTA(P250A) is reduced. This is the first reported evidence that EDEM1 protein recognition might be determined by the structure of the ERAD substrate.
authors.affiliation: Department of Molecular Biology, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland.
authors.forename: Iwona
authors.initials: I
authors.lastname: Sokołowska
authors.forename: Sébastien
authors.initials: S
authors.lastname: Wälchli
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Węgrzyn
authors.forename: Kirsten
authors.initials: K
authors.lastname: Sandvig
authors.forename: Monika
authors.initials: M
authors.lastname: Słomińska-Wojewódzka
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D010947:Plants, Toxic / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D017354:Point Mutation / Q000235:genetics*; D017510:Protein Folding; D021381:Protein Transport / Q000235:genetics; D012276:Ricin / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity*; D013379:Substrate Specificity; D014118:Toxins, Biological / Q000235:genetics / Q000378:metabolism; D014709:Vero Cells
chemical_list: C486654:EDEM1 protein, human; D008565:Membrane Proteins; D014118:Toxins, Biological; D012276:Ricin